Lentivirus-mediated delivery of sonic hedgehog into the striatum stimulates neuroregeneration in a rat model of Parkinson disease.

Parkinson disease (PD) is a progressive neurodegenerative disorder in which the nigrostriatal pathway, consisting of dopaminergic neuronal projections from the substantia nigra to the striatum, degenerates. Viral transduction is currently the most promising in vivo strategy for delivery of therapeutic proteins into the brain for treatment of PD. Sonic hedgehog (Shh) is necessary for cell proliferation, differentiation and neuroprotection in the central nervous system. In this study, we investigated the effects of overexpressed N-terminal product of SHH (SHH-N) in a PD model rat. A lentiviral vector containing SHH-N was stereotactically injected into the striatum 24 h after a striatal 6-OHDA lesion. We found that overexpressed SHH-N attenuated behavioral deficits and reduced the loss of dopamine neurons in the substantia nigra and the loss of dopamine fibers in the striatum. In addition, fluoro-ruby-labeled nigrostriatal projections were also repaired. Together, our results demonstrate the feasibility and efficacy of using the strategy of lentivirus-mediated Shh-N delivery to delay nigrostriatal pathway degeneration. This strategy holds the potential for therapeutic application in the treatment of PD.

The association between sleep deprivation and the risk of cardiovascular diseases: A systematic meta­analysis.

Globally, sleep deprivation is a concerning health issue associated with an increased risk of cardiovascular diseases (CVDs). The present study aimed to explore the association between short-term sleep and the risk of CVDs, taking into consideration sex and age groups. A comprehensive review was conducted by assembling cohort studies that are available in the PubMed, Cochrane Library, and Embase databases. Individuals with ≤5 or ≤6 h of sleep per day were considered as sleep-deprived subjects. To minimize potential bias, two reviewers thoroughly evaluated the selected articles. Relevant data were extracted, and pooled odds ratios (ORs) or relative risks (RRs) were calculated using a random-effects model. In total, 18 cohort studies involving adult subjects were included in the present analysis. The pooled results strongly indicated that sleep deprivation was associated with a greater risk of CVDs [RR: 1.09, 95% confidence interval (CI): 1.02-1.16, P=0.009]. However, when the pooled analysis was stratified by sex and age, the following results were observed: short-term sleep women (RR: 1.06, 95% CI: 0.96-1.17, P=0.27), short-term sleep men (RR: 1.07, 95% CI: 0.97-1.17, P=0.17); ≥18 years-old sleep-deprived population (RR: 1.09, 95% CI: 1.00-1.17, P=0.04), ≥40 years-old sleep-deprived population (RR: 1.09, 95% CI: 0.98-1.22, P=0.11), and subjects with co-existing diseases, such as diabetes and hyperlipidemia (RR: 1.06, 95% CI: 0.94-1.20, P=0.32). In conclusion, short-term sleep is associated with the increased risk of CVDs. Among subjects who were aged ≥18 years-old, there was a strong association with the development of CVDs compared with those who were aged ≥40 years-old. Furthermore, men were at a higher risk of CVDs than women. Adequate sleep (7-8 h per day) may play a role in improving cardiac health. The results of the present study may provide valuable support for further research in public health, highlighting the correlation between sleep deprivation and the risk of CVDs.

The 4b-4c loop of excitatory amino acid transporter 1 containing four critical residues essential for substrate transport.

In the mammalians, the 4b-4c loop of excitatory amino acid transporters (EAATs) spans more than 50 amino-acid residues that are absent in glutamate transporter homologue of Pyrococcus horikoshii (GltPh). This part of insertion is unique for metazoans and indispensable to the localization of EAATs. The excitatory amino acid transporter (EAAT) 1 is one of the two glial glutamate transporters, which are responsible for efficiently clearing glutamate from the synaptic cleft to prevent neurotoxicity and cell death. Although the crystal structure of EAAT1cryst (a human thermostable EAAT1) was resolved in 2017, the structure-function relationship of the 4b-4c loop has not been elucidated in EAAT1cryst. To investigate the role of the 4b-4c loop, we performed alanine-scanning mutagenesis in the mutants and observed dramatically decreased transport activities in T192A, Y194A, N242A, and G245A mutants. The surface expression of T192A and Y194A mutants even decreased by more than 80%, and most of them were detained in the cytoplasm. However, when T192 and Y194 were substituted with conservative residues, the transport activities and the surface expressions of T192S and Y194F were largely recovered, and their kinetic parameters (Km values) were comparable to the wild-type EAAT1 as well. In contrast, N242 and G245 substituted with conservative residues could not rescue the uptake function, suggesting that N242 and G245 may play irreplaceable roles in the glutamate uptake process. These results indicate that the 4b-4c loop of EAAT1 may not only affect the glutamate uptake activity, but also influence the surface localization of EAAT1 by T192 and Y194.Communicated by Ramaswamy H. Sarma.

Glutamate, Glutamate Transporters, and Circadian Rhythm Sleep Disorders in Neurodegenerative Diseases.

Glutamate, a primary excitatory neurotransmitter and an important intermediate in the cellular metabolism of the brain, has a widespread influence in the sleep-wake regulatory system. Glutamate transporters, including vesicular glutamate transporters and excitatory amino acid transporters, serve as the main force controlling the extracellular concentration of glutamate in the brain. These are likely to be critical tools needed for the brain to modulate the sleep-wake cycle and are likely innervated by the circadian rhythm system in a day-night variant pattern. Because in the initial stages, nearly all patients with neurodegenerative diseases have rhythmic sleep disorders that become aggravated with disease development and often exhibit glutamate uptake dysfunction, we examined whether the above glutamate transporters could be used as potential targets to help address circadian rhythm sleep disorders in patients with neurodegenerative diseases. Therefore, in this review, we sought to analyze the principles governing glutamate transmission and discuss whether the circadian rhythm regulatory properties of these processes endow glutamate transporters with unique functions in the sleep-wake shift of the brain. We attempt to provide a theoretical framework in this field for future studies, to help in the exploration of potential therapeutic targets to delay or prevent the development of neurodegenerative diseases.

Paired-Cysteine Scanning Reveals Conformationally Sensitive Proximity between the TM4b-4c Loop and TM8 of the Glutamate Transporter EAAT1.

Excitatory amino acid transporters (EAATs) take up the neurotransmitter glutamate from the synaptic cleft and maintain glutamate concentrations below neurotoxic levels. Recently, the crystal structures of thermostable EAAT1 variants have been reported; however, little is understood regarding the functional mechanism of the transmembrane domain (TM) 4b-4c loop, which contains more than 50 amino acids in mammalian EAATs that are absent in prokaryotic homologues. To explore the spatial position and function of TM4 during the transport cycle, we introduced pairwise cysteine substitutions between the TM4b-4c loop and TM8 in a cysteine-less version of EAAT1, CL-EAAT1. We observed pronounced inhibition of transport by Cu(II)(1,10-phenanthroline)3 (CuPh) for doubly substituted V238C/I469C and A243C/I469C variants, but not for corresponding singly substituted CL-EAAT1 or for more than 20 other double-cysteine variants. Dithiothreitol treatment partially restored the uptake activity of the CuPh-treated V238C/I469C and A243C/I469C doubly substituted variants, confirming that the effects of CuPh on these variants were due to the formation of intramolecular disulfide bonds. Glutamate, KCl, and d,l-threo-ß-benzyloxy-aspartate weakened CuPh inhibition of the V238C/I469C variant, but only KCl weakened CuPh inhibition of the V243C/I469C variant, suggesting that the TM4b-4c loop and TM8 are separated from each other in the inward-facing conformations of EAAT1. Our results suggest that the TM4b-4c loop and TM8 are positioned in close proximity during the transport cycle and are less closely spaced in the inward-facing conformation.

Cloning and bioinformatical analysis of the N-terminus of the sonic hedgehog gene.

The sonic hedgehog protein not only plays a key role in early embryonic development, but also has essential effects on the adult nervous system, including neural stem cell proliferation, differentiation, migration and neuronal axon guidance. The N-terminal fragment of sonic hedgehog is the key functional element in this process. Therefore, this study aimed to clone and analyze the N-terminal fragment of the sonic hedgehog gene. Total RNA was extracted from the notochord of a Sprague-Dawley rat at embryonic day 9 and the N-terminal fragment of sonic hedgehog was amplified by nested reverse transcription-PCR. The N-terminal fragment of the sonic hedgehog gene was successfully cloned. The secondary and tertiary structures of the N-terminal fragment of the sonic hedgehog protein were predicted using Jpred and Phyre online.